Hybrid pumpkin plant named HMX2738

ABSTRACT

A novel hybrid pumpkin plant, designated HMX2738 is disclosed. The invention relates to the seeds of pumpkin hybrid HMX2738, to the plants and plant parts of hybrid pumpkin HMX2738, and to methods for producing a pumpkin plant by crossing the hybrid pumpkin HMX2738 with itself or another pumpkin plant.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive hybrid pumpkin plants, such as hybrid plant designatedHMX2738 and to methods of making and using such hybrids.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Pumpkin is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding pumpkinhybrids that are agronomically sound or unique. The reasons for thisgoal are to maximize the amount of fruit produced on the land used(yield) as well as to improve the fruit appearance, the fruit shape andsize, eating and processing qualities and/or the plant agronomic andhorticultural qualities. To accomplish this goal, the pumpkin breedermust select and develop pumpkin plants that have the traits that resultin superior parental lines that combine to produce superior hybrids.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments there is provided anovel hybrid pumpkin, designated HMX2738.

This invention thus relates to the seeds of hybrid pumpkin designatedHMX2738, to the plants or parts thereof of hybrid pumpkin designatedHMX2738, to plants or parts thereof consisting essentially all of thephysiological and morphological characteristics of hybrid pumpkindesignated HMX2738 or parts thereof, and/or having all the physiologicaland morphological characteristics of hybrid pumpkin designated HMX2738,and/or having one or more or all of the characteristics of hybridpumpkin designated HMX2738 listed in Table 1 including but not limitedto as determined at the 5% significance level when grown in the sameenvironmental conditions, and/or having one or more of the physiologicaland morphological characteristics of hybrid pumpkin designated HMX2738listed in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or having all the physiological and morphological characteristics ofhybrid pumpkin designated HMX2738 listed in Table 1 including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions and/or having all the physiological andmorphological characteristics of hybrid pumpkin designated HMX2738listed in Table 1 when grown in the same environmental conditions. Theinvention also relates to variants, mutants and trivial modifications ofthe seed or plant of hybrid pumpkin designated HMX2738.

Plant parts of the hybrid pumpkin plant of the present invention arealso provided, such as, but not limited to, a scion, a rootstock, afruit, leaf, flower, peduncle, stalk, root, anther cell, pollen or ovuleobtained from the hybrid plant. The present invention provides fruit ofthe hybrid pumpkin of the present invention. Such fruit and partsthereof could be used as fresh products for consumption or in processesresulting in processed products such as food products comprising one ormore harvested part of the hybrid pumpkin designated HMX2738, such asprepared fruit or parts thereof, canned fruit or parts thereof, freezedried or frozen fruit or parts thereof, diced fruits, juice, preparedfruit cuts, canned pumpkin, pastes, sauces, puree and the like. All suchproducts are part of the present invention and the like. The harvestedpart or food product can be or can comprise hybrid pumpkin fruit fromhybrid pumpkin designated HMX2738. The food products might haveundergone one or more processing steps such as, but not limited tocutting, washing, mixing, frizzing, canning, etc. All such products arepart of the present invention. The present invention also provides plantparts or cells, wherein a plant regenerated from said plants parts orcells has one or more, or essentially all of the phenotypic andmorphological characteristics of hybrid pumpkin designated HMX2738, suchas one or more or all of the characteristics of hybrid pumpkindesignated HMX2738, listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from hybrid pumpkin designatedHMX2738 or from a variety that i) is predominantly derived from hybridpumpkin designated HMX2738, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of hybrid pumpkin designated HMX2738; ii) is clearlydistinguishable from hybrid pumpkin designated HMX2738; and iii) exceptfor differences that result from the act of derivation, conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofhybrid pumpkin designated HMX2738. In some embodiments, the tissueculture is capable of regenerating plants consisting essentially all ofthe physiological and morphological characteristics of hybrid pumpkindesignated HMX2738, and/or having all the physiological andmorphological characteristics of hybrid pumpkin designated HMX2738,and/or having the physiological and morphological characteristics ofhybrid pumpkin designated HMX2738, and/or having the characteristics ofhybrid pumpkin designated HMX2738. In one embodiment, the regeneratedplants have the characteristics of hybrid pumpkin designated HMX2738listed in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions.

In some embodiments, the plant parts and cells used to produce suchtissue cultures will be embryos, meristematic cells, seeds, callus,pollen, leaves, anthers, pistils, roots, root tips, stems, petioles,fruits, cotyledons, hypocotyls, ovaries, seed coat, fruits, stalks,endosperm, flowers, axillary buds or the like. Protoplasts produced fromsuch tissue culture are also included in the present invention. Thepumpkin shoots, roots and whole plants regenerated from the tissueculture, as well as the fruit produced by said regenerated plants arealso part of the invention. In some embodiments, the whole plantsregenerated from the tissue culture have one, more than one, or all ofthe physiological and morphological characteristics of pumpkin hybriddesignated HMX2738 listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In the present application, vegetativelypropagating can be interchangeably used with vegetative reproduction. Insome embodiments, the methods comprise collecting a part of a hybridpumpkin designated HMX2738 and regenerating a plant from said part. Insome embodiments, the part can be for example a stem cutting that isrooted into an appropriate medium according to techniques known by theone skilled in the art. Plants, plant parts and fruits thereof producedby such methods are also included in the present invention. In anotheraspect, the plants and fruits thereof produced by such methods consistessentially all of the physiological and morphological characteristicsof hybrid pumpkin designated HMX2738, and/or having all thephysiological and morphological characteristics of hybrid pumpkindesignated HMX2738 and/or having the physiological and morphologicalcharacteristics of hybrid pumpkin designated HMX2738 and/or having thecharacteristics of hybrid pumpkin designated HMX2738. In someembodiments, plants or fruits thereof produced by such methods consistof one, more than one, or all physiological and morphologicalcharacteristics of pumpkin hybrid designated HMX2738 listed in Table 1including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions.

Further included in the invention are methods for producing fruitsand/or seeds from the hybrid pumpkin designated HMX2738. In someembodiments, the methods comprise growing a hybrid pumpkin designatedHMX2738 to produce pumpkin fruits and/or seeds. In some embodiments, themethods further comprise harvesting the hybrid pumpkin fruits and/orseeds. Such fruits and/or seeds are part of the present invention. Insome embodiments, such fruits and/or seeds have all the physiologicaland morphological characteristics of fruit and seed of hybrid pumpkindesignated HMX2738 (e.g. those listed in Table 1) when grown in the sameenvironmental conditions.

Also included in this invention are methods for producing a pumpkinplant. In some embodiments, the pumpkin plant is produced by crossingthe hybrid pumpkin designated HMX2738 with itself or another pumpkinplant. In some embodiments, the other plant can be a pumpkin hybrid orline. When crossed with an inbred line, in some embodiments, a“three-way cross” is produced. When crossed with itself or with another,different hybrid pumpkin, in some embodiments, a “four-way” cross isproduced. Such three and four-way hybrid seeds and plants produced bygrowing said three and four-way hybrid seeds are included in the presentinvention. Methods for producing a three and four-way hybrid pumpkinseed comprising crossing hybrid pumpkin designated HMX2738 pumpkin plantwith a different pumpkin line or hybrid and harvesting the resultanthybrid pumpkin seed are also part of the invention. The hybrid pumpkinseeds produced by the method comprising crossing hybrid pumpkindesignated HMX2738 pumpkin plant with a different pumpkin plant andharvesting the resultant hybrid pumpkin seed are included in theinvention, as are included the hybrid pumpkin plant or parts thereof andseeds produced by said grown hybrid pumpkin plants.

Further included in the invention are methods for producing pumpkinseeds and plants made thereof. In some embodiments, the methods compriseself-pollinating the hybrid pumpkin designated HMX2738 and harvestingthe resultant hybrid seeds. Pumpkin seeds produced by such method arealso part of the invention.

In another embodiment, this invention relates to methods for producing ahybrid pumpkin designated HMX2738 from a collection of seeds. In someembodiments, the collection contains both seeds of inbred parent line(s)of hybrid pumpkin designated HMX2738 seeds and hybrid seeds of HMX2738.Such a collection of seeds might be a commercial bag of seeds. In someembodiments, said methods comprise planting the collection of seeds.When planted, the collection of seeds will produce inbred parent linesof hybrid pumpkin HMX2738 and hybrid plants from the hybrid seeds ofHMX2738. In some embodiments, said inbred parent lines of hybrid pumpkindesignated HMX2738 plants are identified as having a decreased vigorcompared to the other plants (i.e. hybrid plants) grown from thecollection of seeds. In some embodiments, said decreased vigor is due tothe inbreeding depression effect and can be identified for example by aless vigorous appearance for vegetative and/or reproductivecharacteristics including a shorter plant height, small fruit size,fruit shape, fruit color or other characteristics. In some embodiments,seeds of the inbred parent lines of the hybrid pumpkin HMX2738 arecollected and, if new inbred plants thereof are grown and crossed in acontrolled manner with each other, the hybrid pumpkin HMX2738 will berecreated.

This invention also relates to methods for producing other pumpkinplants derived from hybrid pumpkin HMX2738 and to the pumpkin plantsderived by the use of those methods.

In some embodiments, such methods for producing a pumpkin plant derivedfrom the hybrid variety HMX2738 comprise (a) self-pollinating the hybridpumpkin HMX2738 plant at least once to produce a progeny plant derivedfrom pumpkin hybrid HMX2738; In some embodiments, the methods furthercomprise (b) crossing the progeny plant derived from pumpkin hybridHMX2738 with itself or a second pumpkin plant to produce a seed of aprogeny plant of a subsequent generation; In some embodiments, themethods further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the methods further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond pumpkin plant to produce a pumpkin plant further derived from thehybrid pumpkin HMX2738. In further embodiments, steps (b), (c) and/or(d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or moregenerations to produce a pumpkin plant derived from the hybrid pumpkinvariety HMX2738. In some embodiments, within each crossing cycle, thesecond plant is the same plant as the second plant in the last crossingcycle. In some embodiments, within each crossing cycle, the second plantis different from the second plant in the last crossing cycle.

Another method for producing a pumpkin plant derived from the hybridvariety HMX2738, comprises the steps of: (a) crossing the hybrid pumpkinHMX2738 plant with a second pumpkin plant to produce a progeny plantderived from pumpkin hybrid HMX2738; In some embodiments, the methodfurther comprise (b) crossing the progeny plant derived from pumpkinhybrid HMX2738 with itself or a second pumpkin plant to produce a seedof a progeny plant of a subsequent generation; In some embodiments, themethod further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the method further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond pumpkin plant to produce a pumpkin plant derived from the hybridpumpkin variety HMX2738. In a further embodiment, steps (b), (c) and/or(d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or moregenerations to produce a pumpkin plant derived from the hybrid pumpkinvariety HMX2738. In some embodiments, within each crossing cycle, thesecond plant is the same plant as the second plant in the last crossingcycle. In some embodiments, within each crossing cycle, the second plantis different from the second plant in the last crossing cycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the hybrid pumpkinHMX2738 and plants or seeds obtained from such methods. The desiredtrait(s) may be, but not exclusively, a single gene. In someembodiments, the gene is a dominant allele. In some embodiments, thegene is a partially dominant allele. In some embodiments, the gene is arecessive allele. In some embodiments, the gene or genes will confersuch traits, including but not limited to male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal,mycoplasma or viral disease, enhanced plant quality such as improveddrought or salt tolerance, water-stress tolerance, improvedstandability, enhanced plant vigor, improved shelf life, delayedsenescence or controlled ripening, enhanced nutritional quality such asincreased sugar content or increased sweetness, increased texture,flavor and aroma, improved fruit length and/or size, protection forcolor, fruit shape, uniformity, length or diameter, refinement or depth,lodging resistance, yield and recovery, improve fresh cut application,specific aromatic compounds, specific volatiles, flesh texture, specificnutritional components. For the present invention and the skilledartisan, disease is understood to include, but not limited to fungaldiseases, viral diseases, bacterial diseases, mycoplasma diseases, orother plant pathogenic diseases and a disease resistant plant willencompass a plant resistant to fungal, viral, bacterial, mycoplasma, andother plant pathogens. The gene or genes may be naturally occurringpumpkin gene(s), mutant(s), or genes modified through the use of NewBreeding Techniques. In some embodiments, the method for introducing thedesired trait(s) is a backcrossing process making use of a series ofbackcrosses to at least one of the parent lines of hybrid pumpkinHMX2738 during which the desired trait(s) is maintained by selection.The single gene conversion plants that can be obtained by the methodsare included in the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as at least one ofthe parent lines of hybrid pumpkin HMX2738. Alternatively, if the traitis not modified into each newly developed line/cultivar such as at leastone of the parent lines of hybrid pumpkin HMX2738, another typicalmethod used by breeders of ordinary skill in the art to incorporate themodified gene is to take a line already carrying the modified gene andto use such line as a donor line to transfer the modified gene into oneor more of the parents of the newly developed hybrid.

The same would apply for a naturally occurring trait or one arising fromspontaneous or induced mutations.

In some embodiments, the backcross breeding process of hybrid pumpkinHMX2738 comprises (a) crossing one of the parental inbred line plants ofHMX2738 with plants of another line that comprise the desired trait(s)to produce F1 progeny plants In some embodiments, the process furthercomprises (b) selecting the F1 progeny plants that have the desiredtrait(s) In some embodiments, the process further comprises (c) crossingthe selected F1 progeny plants with the parental inbred pumpkin lines ofhybrid HMX2738 plants to produce backcross progeny plants In someembodiments, the process further comprises (d) selecting for backcrossprogeny plants that have the desired trait(s) and physiological andmorphological characteristics of the pumpkin parental inbred line ofhybrid pumpkin HMX2738 to produce selected backcross progeny plants; Insome embodiments, the process further comprises (e) repeating steps (c)and (d) one, two, three, four, five six, seven, eight, nine or moretimes in succession to produce selected, second, third, fourth, fifth,sixth, seventh, eighth, ninth or higher backcross progeny plants thathave the desired trait(s) and otherwise consist essentially all of thephysiological and morphological characteristics of the parental inbredpumpkin line of hybrid pumpkin HMX2738, and/or have the desired trait(s)and otherwise the physiological and morphological characteristics of theparental pumpkin inbred line of hybrid pumpkin HMX2738, and/or have allthe desired trait(s) and otherwise the physiological and morphologicalcharacteristics of the parental inbred pumpkin line of pumpkin hybridHMX2738 as determined in Table 1, including but not limited to whengrown in the same environmental conditions or including but not limitedto at a 5% significance level when grown in the same environmentalconditions. The pumpkin plants or seed produced by the methods are alsopart of the invention, as are the hybrid pumpkin HMX2738 plants thatcomprised the desired trait. Backcrossing breeding methods, well knownto one skilled in the art of plant breeding will be further developed insubsequent parts of the specification.

In an embodiment of this invention is a method of making a backcrossconversion of hybrid pumpkin HMX2738. In some embodiments, the methodcomprises crossing one of the parental pumpkin inbred line plants ofhybrid HMX2738 with a donor plant comprising a mutant gene(s), anaturally occurring gene(s) or a gene(s) and/or sequences modifiedthrough New Breeding Techniques conferring one or more desired trait toproduce F1 progeny plants. In some embodiments, the method furthercomprises selecting an F1 progeny plant comprising the naturallyoccurring gene(s), mutant gene(s) or modified gene(s) and/or sequencesconferring the one or more desired trait; In some embodiments, themethod further comprises backcrossing the selected progeny plant to theparental pumpkin inbred line plants of hybrid HMX2738. This method mayfurther comprise the step of obtaining a molecular marker profile of theparental pumpkin inbred line plants of hybrid HMX2738 and using themolecular marker profile to select for the progeny plant with thedesired trait and the molecular marker profile of the parental pumpkininbred line plants of hybrid HMX2738. In some embodiments, this methodfurther comprises crossing the backcross progeny plant HMX2738containing the naturally occurring gene(s), the mutant gene(s) or themodified gene(s) and or sequences conferring the one or more desiredtrait with the second parental inbred pumpkin line plants of hybridpumpkin HMX2738 in order to produce the hybrid pumpkin HMX2738comprising the naturally occurring gene(s), the mutant gene(s) ormodified gene(s) and/or sequences conferring the one or more desiredtraits. The plants or parts thereof produced by such methods are alsopart of the present invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the parental pumpkin inbred line of hybrid HMX2738 isat least 1, 2, 3, 4, 5, or more.

A single locus may contain several genes. A single locus conversion alsoallows for making one or more site specific changes to the plant genome,such as, without limitation, one or more nucleotide change, deletion,insertions, etc. In some embodiments, the single locus conversion isperformed by genome editing, a.k.a. genome editing with engineerednucleases (GEEN). In some embodiments, the genome editing comprisesusing one or more engineered nucleases. In some embodiments, theengineered nucleases include, but are not limited to Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALEN5), the CRISPR/Cas system, and engineered meganuclease,re-engineered homing endonucleases and endonucleases for DNA guidedgenome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by the personskilled in the art and herein are collectively referred to as “NewBreeding Techniques”. In some embodiments, one or more above-mentionedgenome editing method is directly applied on a plant of the presentinvention, rather than the parental pumpkin inbred lines of hybridHMX2738. Accordingly, a cell containing edited genome, or a plant partcontaining such cell can be isolated and used to regenerate a novelplant which has a new trait conferred by said genome editing, andotherwise all of the physiological and morphological characteristics ofhybrid pumpkin plant HMX2738.

The invention further provides methods for developing pumpkin plants ina pumpkin plant breeding program using plant breeding techniquesincluding but not limited to, recurrent selection, backcrossing,pedigree breeding, genomic selection, molecular marker (IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, Single NucleotidePolymorphism (SNP), etc.) enhanced selection, genetic marker enhancedselection and transformation. Seeds, pumpkin plants, and parts thereofproduced by such breeding methods are also part of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the pumpkin hybrid HMX2738 orinbred parental lines thereof. Variants, mutants and trivialmodifications of the seed or plant of hybrid pumpkin HMX2738 or inbredparental lines thereof can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisenseand RNA interference and other techniques such as the New BreedingTechniques. For more information of mutagenesis in plants, such asagents or protocols, see Acquaah et al. (Principles of plant geneticsand breeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464,which is herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the hybridpumpkin HMX2738 and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newpumpkin plants which comprise one or more or all of the morphologicaland physiological characteristics of hybrid pumpkin HMX2738. In someembodiments, the new pumpkin plants obtained from the screening processcomprise all of the morphological and physiological characteristics ofthe pumpkin hybrid HMX2738 and one or more additional or differentmorphological and physiological characteristics that the pumpkin hybridHMX2738 does not have.

This invention also is directed to methods for producing a pumpkin plantby crossing a first parent pumpkin plant with a second parent pumpkinplant wherein either the first or second parent pumpkin plant is ahybrid pumpkin plant of HMX2738. Further, both first and second parentpumpkin plants can come from the hybrid pumpkin plant HMX2738. Further,the hybrid pumpkin plant HMX2738 can be self-pollinated i.e. the pollenof a hybrid pumpkin plant HMX2738 can pollinate the ovule of the samehybrid pumpkin plant HMX2738. When crossed with another pumpkin plant, ahybrid seed is produced. Such methods of hybridization andself-pollination are well known to those skilled in the art of breeding.

An inbred pumpkin line such as one of the parental lines of hybridpumpkin HMX2738 has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.An inbred line can also be produced though the dihaploid system whichinvolves doubling the chromosomes from a haploid plant or embryo thusresulting in an inbred line that is genetically stable (homozygous) andcan be reproduced without altering the inbred line. Haploid plants couldbe obtained from haploid embryos that might be produced frommicrospores, pollen, anther cultures or ovary cultures or spontaneoushaploidy. The haploid embryos may then be doubled by chemical treatmentssuch as by colchicine or be doubled autonomously. The haploid embryosmay also be grown into haploid plants and treated to induce thechromosome doubling. In either case, fertile homozygous plants areobtained. A hybrid variety is classically created through thefertilization of an ovule from an inbred parental line by the pollen ofanother, different inbred parental line. Due to the homozygous state ofthe inbred line, the produced gametes carry a copy of each parentalchromosome. As both the ovule and the pollen bring a copy of thearrangement and organization of the genes present in the parental lines,the genome of each parental line is present in the resulting F1 hybrid,theoretically in the arrangement and organization created by the plantbreeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga pumpkin plant derived from hybrid pumpkin HMX2738 by crossing hybridpumpkin plant HMX2738 with a second pumpkin plant. In some embodiments,the methods further comprise obtaining a progeny seed from the cross. Insome embodiments, the methods further comprise growing the progeny seed,and possibly repeating the crossing and growing steps with the pumpkinhybrid plant HMX2738-derived plant from 0 to 7 or more times. Thus, anysuch methods using the hybrid pumpkin plant HMX2738 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using hybrid pumpkinplant HMX2738 as a parent are within the scope of this invention,including plants derived from hybrid pumpkin plant HMX2738. In someembodiments, such plants have one, more than one or all physiologicaland morphological characteristics of the pumpkin hybrid plant HMX2738listed in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions. Insome embodiments, such plants might exhibit additional and desiredcharacteristics or traits such as high seed yield, high seedgermination, seedling vigor, early maturity, high fruit yield, ease offruit setting, disease tolerance or resistance, lodging resistance andadaptability for soil and climate conditions. Consumer-driven traits,such as a preference for a given fruit size, fruit shape, fruit color,fruit texture, fruit taste, fruit firmness, fruit sugar content areother traits that may be incorporated into new pumpkin plants developedby this invention.

A pumpkin plant can also be propagated vegetatively. A part of theplant, for example a shoot tissue, is collected, and a new plant isobtained from the part. Such part typically comprises an apical meristemof the plant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots, or is grafted onto a pumpkin plant or a rootstock prepared tosupport growth of shoot tissue. This is achieved using methods wellknown in the art. Accordingly, in one embodiment, a method ofvegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, a fruit isharvested from said plant. In one embodiments, such fruits and plantshave all the physiological and morphological characteristics of hybridpumpkin designated HMX2738 fruits and plants when grown in the sameenvironmental conditions. In one embodiment, the fruit is processed intoproducts such as canned pumpkin fruits and/or parts thereof, freezedried or frozen fruit and/or parts thereof, fresh or prepared fruitand/or parts thereof or pastes, sauces, puree and the like.

The invention is also directed to the use of the hybrid pumpkin plantHMX2738 in a grafting process. In one embodiment, the hybrid pumpkinplant HMX2738 is used as the scion while in another embodiment, thehybrid pumpkin plant HMX2738 is used as a rootstock.

In some embodiments, the present invention teaches a seed of hybridpumpkin designated HMX2738, wherein a representative sample of seed ofsaid hybrid is deposited under NCIMB No. 43504.

In some embodiments, the present invention teaches a pumpkin plant, or apart thereof, produced by growing the deposited HMX2738 seed.

In some embodiments, the present invention teaches pumpkin plant parts,wherein the pumpkin part is selected from the group consisting of: aleaf, a flower, a fruit, a seed, an ovule, pollen, a cell, a rootstock,and a scion.

In some embodiments, the present invention teaches a pumpkin plant, or apart thereof, having all of the characteristics of hybrid HMX2738 aslisted in Table 1 of this application including but not limited to whengrown in the same environmental conditions.

In some embodiments, the present invention teaches a pumpkin plant, or apart thereof, having all of the physiological and morphologicalcharacteristics of hybrid HMX2738, wherein a representative sample ofseed of said hybrid was deposited under NCIMB No. 43504.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited HMX2738 seed, wherein cells of the tissue culture are producedfrom a plant part selected from the group consisting of protoplasts,embryos, meristematic cells, callus, pollen, ovules, flowers, seeds,leaves, roots, root tips, anthers, stems, petioles, fruits, axillarybuds, cotyledons and hypocotyls. In some embodiments, the plant partincludes protoplasts produced from a plant grown from the depositedHMX2738 seed.

In some embodiments, the present invention teaches a compositioncomprising regenerable cells produced from the plant or plant part grownfrom the deposited hybrid HMX2738 seed, or other plant part or plantcell. In some embodiments, the composition comprises a growth media. Insome embodiments, the growth media is solid or a synthetic cultivationmedium. In some embodiments, the composition is a pumpkin plantregenerated from the tissue culture from a plant grown from thedeposited HMX2738 seed, said plant having the characteristics of hybridHMX2738, wherein a representative sample of seed of said hybrid isdeposited under NCIMB No. 43504.

In some embodiments, the present invention teaches a pumpkin fruitproduced from the plant grown from the deposited HMX2738 seed. In oneembodiments, such fruits have all the physiological and morphologicalcharacteristics of hybrid pumpkin designated HMX2738 fruits when grownin the same environmental conditions.

In some embodiments, methods of producing said pumpkin fruit comprise a)growing the pumpkin plant from deposited HMX2738 seed to produce apumpkin fruit, and b) harvesting said pumpkin fruit. In someembodiments, the present invention also teaches a pumpkin fruit producedby the method of producing pumpkin fruit and/or seed as described above.In one embodiments, such fruits have all the physiological andmorphological characteristics of fruits of hybrid pumpkin designatedHMX2738 (e.g. those listed in Table 1) when grown in the sameenvironmental conditions.

In some embodiments, the present invention teaches methods for producinga pumpkin seed comprising crossing a first parent pumpkin plant with asecond parent pumpkin plant and harvesting the resultant pumpkin seed,wherein said first parent pumpkin plant and/or second parent pumpkinplant is the pumpkin plant produced from the deposited HMX2738 seed or apumpkin plant having all of the characteristics of pumpkin hybridHMX2738 as listed in Table 1 including but not limited to when grown inthe same environmental conditions.

In some embodiments, the present invention teaches methods for producinga pumpkin seed comprising self-pollinating the pumpkin plant grown fromthe deposited HMX2738 seed and harvesting the resultant pumpkin seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the pumpkin plant grown from the depositedHMX2738 seed, said method comprising a) collecting part of a plant grownfrom the deposited HMX2738 seed and b) regenerating a plant from saidpart.

In some embodiments, the method further comprises harvesting a fruitand/or seed from said vegetatively propagated plant.

In some embodiments, the present invention teaches the plant and thefruit and/or seed of plants vegetatively propagated from plant parts ofplants grown from the deposited HMX2738 seed. In one embodiments, suchplant, fruits and/or seeds have all the physiological and morphologicalcharacteristics of hybrid pumpkin designated HMX2738 plant, fruitsand/or seeds of pumpkin hybrid HMX2738 (e.g. those listed in Table 1)when grown in the same environmental conditions.

In some embodiments, the present invention teaches methods of producinga pumpkin plant derived from the hybrid variety HMX2738. In someembodiment the methods comprise (a) self-pollinating the plant grownfrom the deposited HMX2738 seed at least once to produce a progeny plantderived from pumpkin hybrid HMX2738. In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from pumpkinhybrid HMX2738 with itself or a second pumpkin plant to produce a seedof a progeny plant of a subsequent generation; and; (c) growing theprogeny plant of the subsequent generation from the seed, and crossingthe progeny plant of the subsequent generation with itself or a secondpumpkin plant to produce a pumpkin plant derived from the hybrid pumpkinvariety HMX2738. In some embodiments said methods further comprise thestep of: (d) repeating steps b) and/or c) for at least 1, 2, 3, 4, 5, 6,7, or more generation to produce a pumpkin plant derived from the hybridpumpkin variety HMX2738.

In some embodiments, the present invention teaches methods of producinga pumpkin plant derived from the hybrid variety HMX2738, the methodscomprising (a) crossing the plant grown from the deposited HMX2738 seedwith a second pumpkin plant to produce a progeny plant derived frompumpkin hybrid HMX2738. In some embodiments, the method furthercomprises; (b) crossing the progeny plant derived from pumpkin hybridHMX2738 with itself or a second pumpkin plant to produce a seed of aprogeny plant of a subsequent generation; and; (c) growing the progenyplant of the subsequent generation from the seed; (d) crossing theprogeny plant of the subsequent generation with itself or a secondpumpkin plant to produce a pumpkin plant derived from the hybrid pumpkinvariety HMX2738. In some embodiments said methods further comprise thesteps of: (e) repeating step (b), (c) and/or (d) for at least 1, 2, 3,4, 5, 6, 7 or more generation to produce a pumpkin plant derived fromthe hybrid pumpkin variety HMX2738.

In some embodiments, the present invention teaches plants grown from thedeposited HMX2738 seed wherein said plants comprise a single locusconversion. As used herein, the term “a” or “an” refers to one or moreof that entity; for example, “a single locus conversion” refers to oneor more single locus conversions or at least one single locusconversion. As such, the terms “a” (or “an”), “one or more” and “atleast one” are used interchangeably herein. In addition, reference to“an element” by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.In some embodiments said single locus conversion confers said plantswith a trait selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, enhanced plant qualitysuch as improved drought or salt tolerance, water stress tolerance,improved standability, enhanced plant vigor, improved shelf life,delayed senescence or controlled ripening, increased nutritional qualitysuch as increased sugar content or increased sweetness, increasedtexture, flavor and aroma, improved fruit length and/or size, protectionfor color, fruit shape, uniformity, length or diameter, refinement ordepth lodging resistance, yield and recovery when compared to a suitablecheck plant. In some embodiments, the check plant is a pumpkin hybridHMX2738 not having said single locus conversion. In some embodiments,the at least one single locus conversion is an artificially mutated geneor a gene or nucleotide sequence modified through the use of NewBreeding Techniques.

In some embodiments, the present invention provides a method ofproducing a commodity plant product comprising collecting the commodityplant product from the plant of the present invention. The commodityplant product produced by said method is also part of the presentinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Adaptability. A plant that has adaptability is a plant able to grow wellin different growing conditions (climate, soils, etc.).

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Commodity plant product. A “commodity plant product” refers to anycomposition or product that is comprised of material derived from aplant, seed, plant cell, or plant part of the present invention.Commodity plant products may be sold to consumers and can be viable ornonviable. Nonviable commodity products include but are not limited tononviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, paper, tea, coffee, silage, crushed of whole grain, and any otherfood for human or animal consumption; and biomasses and fuel products;and raw material in industry.

Collection of seeds. In the context of the present invention acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example hybrid seeds having the inbred line of theinvention as a parental line, but that may also contain, mixed togetherwith this first kind of seeds, a second, different kind of seeds, of oneof the inbred parent lines, for example the inbred line of the presentinvention. A commercial bag of hybrid seeds having the inbred line ofthe invention as a parental line and containing also the inbred lineseeds of the invention would be, for example such a collection of seeds.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small fruit size, fewer fruit or othercharacteristics.

Earliness. The earliness relates the number of days from seeding toharvest.

Easy to pick fruit. A fruit that is easy to pick is a fruit that easilydetaches from the plant. Once grabbed and twisted, the fruit will breakbetween the peduncle and the stem. For fruits not easy to pick, thepeduncle breaks off the fruits. A fruit that is easy to pick is also afruit that is easily accessible for harvest. When plants have an openplant habit, the fruits are harvested more easily than when the plantshave closed habit.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Extended harvest. An extended harvest is a plant that produces fruitsthroughout the harvest season.

Field holding ability: Field holding ability is the ability for fruitquality to maintain even after fruit is ripe.

Firm Fruit Exterior. Fruit Firmness subjectively tested under fieldconditions for resistance of fruit exterior against a given pressure.Range is soft, medium, firm and very firm and hard shell.

Flesh color: In the context of the present invention, the flesh color isthe color of the pumpkin flesh.

Grafting. Grafting is the operation by which a rootstock is grafted witha scion. The primary motive for grafting is to avoid damages bysoil-born pest and pathogens when genetic or chemical approaches fordisease management are not available. Grafting a susceptible scion ontoa resistant rootstock can provide a resistant cultivar without the needto breed the resistance into the cultivar. In addition, grafting mayenhance tolerance to abiotic stress, increase yield and result in moreefficient water and nutrient uses.

Immunity to disease(s) and or insect(s). A pumpkin plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Industrial usage. The industrial usage of the pumpkin of the presentinvention comprises the use of the pumpkin fruit for consumption,whether as fresh products or in canning, freezing or any otherindustries.

Intermediate resistance to disease(s) and or insect(s). A pumpkin plantthat restricts the growth and development of specific disease(s) and orinsect(s), but may exhibit a greater range of symptoms or damagecompared to a resistant plants. Intermediate resistant plants willusually show less severe symptoms or damage than susceptible plantvarieties when grown under similar environmental conditions and/orspecific disease(s) and or insect(s) pressure, but may have heavy damageunder heavy pressure. Intermediate resistant pumpkin plants are notimmune to the disease(s) and or insect(s).

Maturity. In the region of best adaptability, maturity is the number ofdays from transplanting to optimal time for fruit harvest.

New Breeding Techniques: New breeding techniques (NBTs) are said ofvarious new technologies developed and/or used to create newcharacteristics in plants through genetic variation, the aim beingtargeted mutagenesis, targeted introduction of new genes or genesilencing (RdDM). The following breeding techniques are within the scopeof NBTs: targeted sequence changes facilitated thru the use of Zincfinger nuclease (ZFN) technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Pat.No. 9,145,565, incorporated by reference in its entirety),Oligonucleotide directed mutagenesis (ODM, a.k.a., site-directedmutagenesis), Cisgenesis and intragenesis, epigenetic approaches such asRNA-dependent DNA methylation (RdDM, which does not necessarily changenucleotide sequence but can change the biological activity of thesequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration for transient gene expression (agro-infiltration“sensu stricto”, agro-inoculation, floral dip), TranscriptionActivator-Like Effector Nucleases (TALEN5, see U.S. Pat. Nos. 8,586,363and 9,181,535, incorporated by reference in their entireties), theCRISPR/Cas system (see U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965;8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814;8,945,839; 8,993,233; and 8,999,641, which are all hereby incorporatedby reference), engineered meganuclease, re-engineered homingendonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics). A major part of today'stargeted genome editing, another designation for New BreedingTechniques, is the applications to induce a DNA double strand break(DSB) at a selected location in the genome where the modification isintended. Directed repair of the DSB allows for targeted genome editing.Such applications can be utilized to generate mutations (e.g., targetedmutations or precise native gene editing) as well as precise insertionof genes (e.g., cisgenes, intragenes, or transgenes). The applicationsleading to mutations are often identified as site-directed nuclease(SDN) technology, such as SDN1, SDN2 and SDN3. For SDN1, the outcome isa targeted, non-specific genetic deletion mutation: the position of theDNA DSB is precisely selected, but the DNA repair by the host cell israndom and results in small nucleotide deletions, additions orsubstitutions. For SDN2, a SDN is used to generate a targeted DSB and aDNA repair template (a short DNA sequence identical to the targeted DSBDNA sequence except for one or a few nucleotide changes) is used torepair the DSB: this results in a targeted and predetermined pointmutation in the desired gene of interest. As to the SDN3, the SDN isused along with a DNA repair template that contains new DNA sequence(e.g. gene). The outcome of the technology would be the integration ofthat DNA sequence into the plant genome. The most likely applicationillustrating the use of SDN3 would be the insertion of cisgenic,intragenic, or transgenic expression cassettes at a selected genomelocation. A complete description of each of these techniques can befound in the report made by the Joint Research Center (JRC) Institutefor Prospective Technological Studies of the European Commission in 2011and titled “New plant breeding techniques—State-of-the-art and prospectsfor commercial development”, which is incorporated by reference in itsentirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture, or incorporated in a plant or plantpart.

Plant height. Plant height is taken from the top of the soil to the topnode of the plant and is measured in centimeters or inches.

Plant Part. As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which pumpkin plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, fruit, rootstock, scions, stems, roots, anthers, pistils, roottips, leaves, meristematic cells, axillary buds, hypocotyls cotyledons,ovaries, seed coat endosperm and the like. In some embodiments, theplant part at least comprises at least one cell of said plant. In someembodiments, the plant part is further defined as a pollen, a meristem,a cell or an ovule. In some embodiments, a plant regenerated from theplant part has all of the phenotypic and morphological characteristicsof a pumpkin hybrid of the present invention, including but not limitedto as determined at the 5% significance level when grown in the sameenvironmental conditions.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A pumpkin plant thatrestricts the growth and development of specific disease(s) and orinsect(s) under normal disease(s) and or insect(s) attack pressure whencompared to susceptible plants. These pumpkin plants can exhibit somesymptoms or damage under heavy disease(s) and or insect(s) pressure.Resistant pumpkin plants are not immune to the disease(s) and orinsect(s).

Rootstock. A rootstock is the lower part of a plant capable of receivinga scion in a grafting process.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Scion. A scion is the higher part of a plant capable of being graftedonto a rootstock in a grafting process.

Season maturity. Maturity is considered the date of the onset of harvestand is Very Early, Early, Mid Early, Main and Late.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence muted or engineered through the New BreedingTechniques.

Susceptible to disease(s) and or insect(s). A pumpkin plant that issusceptible to disease(s) and or insect(s) is defined as a pumpkin plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A pumpkin plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Yield (Unit Volume/Acre). The yield in units/acre is the actual yield ofthe pumpkin at harvest.

Pumpkin Plants

Practically speaking, all cultivated forms of ornamental or Halloweenpumpkin belong to genus Cucurbita that is grown for its ornamentalfruit. As a crop, pumpkins are grown commercially almost exclusively inthe United States wherever environmental conditions permit theproduction of an economically viable yield. They are harvested by hand.Pumpkins usually develop a running vine on the soil, but many of today'spumpkins have been developed in the form of a compact bush, making themeasier to grow in smaller spaces. On healthy pumpkin plants, there is acanopy of large, reniform and serrated leaves, which may be withoutlobes or with very deep ones. Fruit flesh can be of various shades ofyellow, or even from white to orange. The fruits may have a soft or ahard shell with usually orange, but possibly other, colors and variouspatterns. Pumpkins show a great variety of sizes from small to large andexterior shell colors from uniform to variegated. The flesh can rangefrom white to yellow and, contrary to the winter squash that has afinely textured flesh, usually has a coarse or stringy flesh. In theUnited States, the principal pumpkin growing regions are California,Michigan, Ohio, Illinois, Pennsylvania, New York, and Texas whichproduce approximately 44,000 acres out of a total annual acreage of morethan 74,000 acres (USDA, 2000; ERS 1997), but pumpkins are grown in atleast a small scale in most States and Canada. Pumpkins are available inthe United States in the fall during September and October inassociation with the Halloween holiday at the end of October. Pumpkinsare edible but are usually used for ornamental purposes. The most commonfood use is in pumpkin pies, but use is limited to a few varieties aswinter squash types have more acceptable flesh quality.

Cucurbita pepo is a member of the family Cucurbitaceae. TheCucurbitaceae is a family of about 90 genera and 700 to 760 species,mostly of the tropics. The family includes pumpkins, squash, gourds,watermelon, loofah and several weeds. The genus Cucurbita, to which thepumpkin belongs, includes four major species, pepo, argyrosperma,moschata, and maxima, one minor species, ficifolia and additional wildspecies. Cross-pollination is near complete among the differentCucurbita species. This offers breeders a great potential forinter-specific crosses using conventional breeding procedures andtransferring traits of interest from one to another species. One wouldfor example consider the transfer of Tomato Leaf Curl New Delhi virusresistance from C. moschata into C. pepo as described in WO2017081205.While transferred into summer squash C. pepo rather than winter squashC. pepo pumpkin, it clearly demonstrates the possibility ofinterspecific crosses to transfer genes of interest between cucurbitaspecies. Other crosses could be between moschata and maxima, maxima andpepo, argyrosperma and maxima, directly or indirectly for example thanksto crosses using bridge species such as indeed argyrosperma. Cucurbitapepo L. refers to what is commonly known as the summer squash such asscallop, zucchini, straightneck and crookneck types and winter squashsuch as acorn and pumpkin. The term pumpkin itself has a rather broadmeaning. Generally, it can be said that if the plant produces fruits tobe harvested in a mature stage and are used for pies, jack-o'-lanterns,or stock feed, they are called pumpkins in the U.S. For the purpose ofthe present application, pumpkin is considered as Cucurbita pepo even ifcomprising traits of interest originating and transferred from othercucurbita species.

Cucurbita pepo is a simple diploid species with twenty pairs of highlydifferentiated chromosomes. The plants are monoecious, with separatefemale and male flowers on the same plant. Usually the first few flowersproduced are male, followed by interspersed male and female flowers.Male flowers have 3-5 erect stamens bunched within the corolla of 5fused petals. Female flowers have 3 spreading stigma lobes and animmature fruit (ovary) below the perianth. The spiny, sticky pollenrequires insects for pollination. The primary pollinators are bees,particularly honey bees. Pollination generally occurs in the morningafter the flowers open. Hybrid vigor has been documented in pumpkins andhybrids are gaining more and more popularity amongst farmers withuniformity of plant characteristics.

Hybrid commercial pumpkins seed is produced by hand pollination. Pollenof the male parent is harvested and manually applied to the stigmaticsurface of the female inbred. Prior to and after hand pollination,flowers are covered so that insects do not bring foreign pollen andcreate a mix or impurity. Flowers are tagged to identify pollinatedfruit from which seed will be harvested.

Hybrid commercial pumpkin seed could also be produced through chemicalmethods: in such methods a growth hormone such as Florel is used on thefemale inbred plant so that it produce female flowers only. Bees arethen used to cross pollinate the production field, whereas, only fruitsfrom the inbred plants that have been treated with the growth hormoneare harvested in order to collect the hybrid seeds contained therein.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In pumpkins, these important traits may include higher yield, fieldperformance, fruit and agronomic quality such as smoothness, color,shape and size, flesh color, texture or thickness, leaf shape and size,vine length and branching pattern, resistance to diseases and insects,tolerance to drought and heat, plant habit and size, easier harvestability, less need for fertilizers, peduncle size and color, betteragronomic quality, shell hardness and seed quality. With mechanicalharvesting of many crops, uniformity of plant characteristics such asgermination and stand establishment, growth rate, maturity and plantheight is important.

Particularly desirable traits that may be incorporated by this inventionare improved resistance to different viral, fungal, and bacterialpathogens. Important diseases include but are not limited to PowderyMildew (Podosphaera xanthii) from C. okeechobeensis, Zucchini yellowMosaic virus (ZYMV) from C. moschata, Squash Leaf Curl Virus (SLCV) fromC. moschata, etc. Improved resistance to insect pests is anotherdesirable trait that may be incorporated into new pumpkin plantsdeveloped by this invention. Insect pests affecting the various speciesof pumpkin include Cucumber Beetles, Squash Vine Borer, aphids,whiteflies, spider mites and others.

Other desirable traits include traits related to improved pumpkinfruits. A non-limiting list of fruit phenotypes used during breedingselection include:

-   -   Fruit Size: In pumpkin there is a continuous range of size        (weight) from less than one pound for the smallest to 60 or more        for the largest. Fruits of a related species, Cucurbita maxima,        also called pumpkin, can be as large as 2500 pounds. In general,        Mini pumpkins weigh up to 2 lbs., Small, 3 to 5 lbs., Medium, 10        to 20 lbs., Large 25 lbs., Very large and Giant are above 25        lbs.    -   Fruit shape: Fruit shape means the conformation of the fruit and        generally runs from very flat (height to width ratio less than        0.5) to very tall (height to width ratio greater than 1.5).    -   Fruit color: Fruit color is judged at physiological maturity.        Fruit Color ranges from very pale orange to very dark orange and        can include white, tan, grey, green and striped, mixed or        mottled colors.    -   Ribbing. Ribbing means the longitudinal suture or crease in the        exterior shell of the pumpkin fruit. Ribbing can range from no        ribbing or smooth to very deep ribs.    -   Handle size: Formally called the peduncle (fruit stalk), it is        also known as the fruit stem. Handle Size can be measured in        length and width and can be very small to very large. The Handle        size is an important marketing feature of a Halloween pumpkin. A        Halloween pumpkin with a relative handle size of medium to large        is generally preferred.        Pumpkin Breeding

The goal of pumpkin breeding is to develop new, unique and superiorpumpkin inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing,crossing and selection, producing many new genetic combinations. Suchmaterial could be C. pepo material, but it could also be C.argyrosperma, C. moschata, or C. maxima when such harbor any traits ofinterest to be transferred into the C. pepo pumpkin plant to bedeveloped. Another method used to develop new, unique and superiorpumpkin inbred lines and hybrids occurs when the breeder selects andcrosses two or more parental lines followed by haploid induction andchromosome doubling that result in the development of dihaploid inbredlines. The breeder can theoretically generate billions of differentgenetic combinations via crossing, selfing and mutations and the same istrue for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred linesdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large research monies to develop superior new pumpkininbred lines and hybrids.

The development of commercial pumpkin hybrids requires the developmentof homozygous inbred lines, the crossing of these lines, and theevaluation of the hybrid crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine desirable traits from two or more inbred lines or variousbroad-based sources into breeding pools from which inbred lines aredeveloped by selfing and selection of desired phenotypes or through thedihaploid breeding method followed by the selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i. Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potential use asparents of new hybrid cultivars. Similarly, the development of newinbred lines through the dihaploid system requires the selection of thebest inbreds followed by two to five years of testing in hybridcombinations in replicated plots.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each fruit by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

ii. Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype recurrent parent andthe trait of interest from the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

When the term hybrid pumpkin plant is used in the context of the presentinvention, this also includes any hybrid pumpkin plant where one or moredesired trait has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one, a mutant, a transgenic one or agene or a nucleotide sequence modified by the use of New BreedingTechniques. Backcrossing methods can be used with the present inventionto improve or introduce one or more characteristic into the inbredparental line, thus potentially introducing these traits in to thehybrid pumpkin plant of the present invention. The term “backcrossing”as used herein refers to the repeated crossing of a hybrid progeny backto the recurrent parent, i.e., backcrossing one, two, three, four, five,six, seven, eight, nine, or more times to the recurrent parent. Theparental pumpkin plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental pumpkin plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol.

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a pumpkin plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as pumpkin leaf curl virusresistance in pumpkin, requires selfing the progeny or using molecularmarkers to determine which plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridpumpkin plant according to the invention but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include but are not limited to,resistance for bacterial, fungal, or viral disease (gene Zym-0 forresistance to Zucchini Yellow Mosaic Virus (ZYMV)), agronomic traits,such as leaf silvering and fruit color, such as green, yellow, white orgrey. These genes are generally inherited through the nucleus.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc., Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly oressentially the same adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because a similar variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will betheoretically modified only with regards to genes being transferred,which are maintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred or when usingmolecular markers that can identify the trait of interest.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii. Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagated by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagated as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by intercrossing a number of genotypesselected for good combining ability in all possible hybrid combinations,with subsequent maintenance of the variety by open pollination. Whetherparents are (more or less inbred) seed-propagated lines, as in somesugar beet and beans (Vicia) or clones, as in herbage grasses, cloversand alfalfa, makes no difference in principle. Parents are selected ongeneral combining ability, sometimes by test crosses or toperosses, moregenerally by polycrosses. Parental seed lines may be deliberately inbred(e.g. by selfing or sib crossing). However, even if the parents are notdeliberately inbred, selection within lines during line maintenance willensure that some inbreeding occurs. Clonal parents will, of course,remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflowerbroccoli and tomato. Hybrids can be formed in a number of differentways, including by crossing two parents directly (single cross hybrids),by crossing a single cross hybrid with another parent (three-way ortriple cross hybrids), or by crossing two different hybrids (four-way ordouble cross hybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial pumpkin seed is produced by controlled handpollination. The male flowers from the male plants are harvested andused to pollinate the stigmatic surface of the female flowers on thefemale plants. Prior to, and after hand pollination, flowers are coveredso that insects do not bring foreign pollen and create a mix orimpurity. Flowers are tagged to identify pollinated fruit from whichseed will be harvested.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. The male parent may be plantedat the top of the field for efficient male flower collection duringpollination. Pollination is started when the female parent flower isready to be fertilized. Female flower buds that are ready to open in thefollowing days are identified, covered with paper cups or small paperbags that prevent bee or any other insect from visiting the femaleflowers, and marked with any kind of material that can be easily seenthe next morning. In some embodiments, this process is best done in theafternoon. The male flowers of the male parent are collected in theearly morning before they are open and visited by pollinating insects.The covered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked fruits are harvested. In some embodiments, the malepollen used for fertilization has been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Breeding of dioeciousspecies can also be done by growing equal amount of each parent plant.Insects are placed in the field or greenhouses for transfer of pollenfrom the male parent to the female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant lines. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke a shape change inthe double strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

ix. Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into pumpkin plants. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means or mutating agentsincluding temperature, long-term seed storage, tissue cultureconditions, radiation (such as X-rays, Gamma rays, neutrons, Betaradiation, or ultraviolet radiation), chemical mutagens (such as baseanalogs like 5-bromo-uracil), antibiotics, alkylating agents (such assulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acidor acridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in W. R.Fehr, 1993, Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intopumpkin varieties.

x. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossing is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol.109, pg. 4227-4232; Zhang et al., 2008 Plant Cell Rep. December 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg. 63-72; Doubled Haploid Production in CropPlants 2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform inbred lines and isespecially desirable as an alternative to sexual inbreeding oflonger-generation crops. By producing doubled haploid progeny, thenumber of possible gene combinations for inherited traits is moremanageable. Thus, an efficient doubled haploid technology cansignificantly reduce the time and the cost of inbred and cultivardevelopment.

xi. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xii. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryos fromcrosses to rapidly move to the next generation of backcrossing orselfing or wherein plants fail to produce viable seed. In this process,the fertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In Vitro Culture of Higher Plants,Springer, ISBN 079235267, 9780792352679, which is incorporated herein byreference in its entirety).

Grafting

Grafting is a process that has been used for many years in crops such ascucurbitaceae, but only more recently for some commercial watermelonproduction. Grafting may be used to provide a certain level ofresistance to telluric pathogens such as Phytophthora or to certainnematodes. Grafting is therefore intended to prevent contact between theplant or variety to be cultivated and the infested soil. The variety ofinterest used as the graft or scion, optionally an F1 hybrid, is graftedonto the resistant plant used as the rootstock. The resistant rootstockremains healthy and provides, from the soils, the normal supply for thegraft that it isolates from the diseases. In some recent developments,it has also been shown that some rootstocks are also able to improve theagronomic value for the grafted plant and in particular the equilibriumbetween the vegetative and generative development that are alwaysdifficult to balance in pumpkin cultivation.

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection or in a backcross program to improvethe parent lines for a specific trait.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, vigor, plant health, maturity, branching, plantheight, leaf coverage, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (fruit) orbiomass production; effects on plant growth that results in an increasedseed yield for a crop; effects on plant growth which result in anincreased yield; effects on plant growth that lead to an increasedresistance or tolerance to disease including fungal, viral or bacterialdiseases, to mycoplasma, or to pests such as insects, mites or nematodesin which damage is measured by decreased foliar symptoms such as theincidence of bacterial or fungal lesions, or area of damaged foliage orreduction in the numbers of nematode cysts or galls on plant roots, orimprovements in plant yield in the presence of such plant pests anddiseases; effects on plant growth that lead to increased metaboliteyields; effects on plant growth that lead to improved aesthetic appealwhich may be particularly important in plants grown for their form,color, for example the color intensity of pumpkin exocarp (skin) of saidfruit.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Transcriptome Sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, immune labeling, immunosorbent electron microscopy (ISEM),and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene, or QTL or a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the mRNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. Furthermore, thespecificity of the assay is mainly determined by the primers, which cangive false-positive results. However, the most important issueconcerning conventional RT-PCR is the fact that it is a semi or even alow quantitative technique, where the amplicon can be visualized onlyafter the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general nonspecific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al., 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg.169-200; Mardis 2008 Genomics and Human Genetics vol. 9 pg. 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line havingcertain favorable traits for commercial production. In one embodiment,the elite line may contain other genes that also impart said line withthe desired phenotype. When crossed together, the donor and recipientplant may create a progeny plant with combined desirable loci which mayprovide quantitatively additive effect of a particular characteristic.In that case, QTL mapping can be involved to facilitate the breedingprocess.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait. Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that is associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow do those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R. G. Landes, Austin). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of pumpkin can be usedfor the in vitro regeneration of pumpkin plants. Tissues cultures ofvarious tissues of pumpkin and regeneration of plants therefrom are wellknown and published. By way of example, a tissue culture comprisingorgans has been used to produce regenerated plants as described inGirish-Chandel et al., Advances in Plant Sciences. 2000, 13: 1, 11-17,Costa et al., Plant Cell Report. 2000, 19: 3327-332, Plastira et al.,Acta Horticulturae. 1997, 447, 231-234, Zagorska et al., Plant CellReport. 1998, 17: 12 968-973, Asahura et al., Breeding Science. 1995,45: 455-459, Chen et al., Breeding Science. 1994, 44: 3, 257-262, Patilet al., Plant and Tissue and Organ Culture. 1994, 36: 2,255-258. It isclear from the literature that the state of the art is such that thesemethods of obtaining plants are routinely used and have a very high rateof success. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce pumpkin plants having thephysiological and morphological characteristics of hybrid pumpkin plantHMX2738.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of New HMX2738 Pumpkin Variety

Breeding History:

Pumpkin hybrid plant HMX2738 has superior characteristics. The female(FPUMP1) and male (MPUMP1) parents were crossed to produce hybrid (F1)seeds of HMX2738. The seeds of HMX2738 can be grown to produce hybridplants and parts thereof. The hybrid HMX2738 can be propagated by seedsproduced from crossing pumpkin inbred line FPUMP1 with pumpkin inbredline MPUMP1 or vegetatively.

The origin and breeding history of hybrid plant HMX2738 can besummarized as follows: the line FPUMP1 (a proprietary line owned byHM.CLAUSE, Inc.) was used as the female plant and crossed by pollen fromMPUMP1 (a proprietary line licensed by HM.CLAUSE, Inc.). The first trialplanting of this hybrid was done in Davis, Calif. in the summer of 2013.The hybrid was further trialed for four years, an example of such trialsbeing disclosed in Tables 2 and 3.

The inbred line FPUMP1 is used as the female parent in this cross.FPUMP1 can be characterized as a powdery mildew resistant bush plantwith medium size leaves and small size, flat fruit. The fruit maturefrom green to orange and have a hard shell with numerous, prominentwarts.

The inbred line MPUMP1 is used as the male parent in this cross. MPUMP1can be characterized as a large vining plant with large leaves andmedium size, round, white fruit. The fruit have deep furrows.

Pumpkin hybrid plant HMX2738 is similar to pumpkin hybrid plant Racer.Racer is a commercial variety. While similar to pumpkin hybrid plantRacer, there are significant differences including the skin color whichis cream for HMX2738, while Racer is orange. The texture of fruitsurface for HMX2738 is warted while Racer is smooth. The staminateflower sepal length is shorter for HMX2738 at 17.8 mm compared to Racerat 29.3 mm. HMX2738 has a handle length of 7.1 cm while it is 9.1 cm forRacer. The flesh thickness at the blossom end is thicker for HMX2738 at3.4 cm compared to Racer at 2.8 cm.

Some of the criteria used to select the hybrid HMX2738 as well as theinbred parent lines in various generations include: bush plant habit,strong growth, powdery mildew resistance, earliness, large well-attachedhandle, numerous well-defined warts, and white mature fruit color.

The pumpkin hybrid plant HMX2738 has shown uniformity and stability forthe traits, within the limits of environmental influence, as describedin the following Variety Descriptive Information. No variant traits havebeen observed or are expected for agronomical important traits inpumpkin hybrid HMX2738.

Pumpkin hybrid plant HMX2738 has the following morphologic and othercharacteristics, as compared to Racer (based primarily on data collectedin Davis (CA); all experiments done under the direct supervision of theapplicant).

TABLE 1 Trait Scale HMX2738 Racer Plant Disease resistance (PM) absent,present Disease resistance (ZYMV) absent, present Internode lengthcentimeters 12.5 10.6 Spines pilose, prickly, glabrous prickly pricklyLeaf Leaf blade form weakly, medium, or weakly lobed heavily lobedheavily lobed Leaf width centimeters 34.5 38.0 Leaf length centimeters23.5 30.0 Petiole length centimeters 40.0 73.0 Flowers Pistillate flowerpedicel length centimeters 3.8 1.9 Pistillate flower sepal widthmillimeters 2.0 1.8 Pistillate flower sepal length millimeters 11.5 18.5Node number of 1st pistillate count 9.2 10.7 flower Staminate flowerpedicel centimeters 30.1 30.5 length Staminate flower sepal widthmillimeters 2.3 2.5 Staminate flower sepal length millimeters 17.8 29.3Mature fruit Weight kilograms 6.9 5.0 Height centimeters 23.8 20.4 Widthcentimeters 26.1 25.0 Skin color white, cream, yellow, cream orangeorange, red, pink, green, grey green, grey Skin color (RHS) RoyalHorticultural Yellow Group Orange Group Society Colour Charts 11A 25ATexture of surface smooth, rough, warted, warted smooth bullate Grooves(ribs) absent, present present present Distance between groovescentimeters 3.6 4.9 Depth of grooves shallow, medium, deep very shallowshallow Handle length centimeters 7.1 9.1 Handle color RoyalHorticultural Green Group Greyed Yellow Society Colour Charts N137AGroup 151D Handle thickness centimeters 6.4 6.3 Handle detaches easy,medium, difficult difficult difficult Warts absent, present presentabsent Width stem end centimeters 14.3 12.9 Width blossom endcentimeters 12.8 13.9 Diameter at broadest point centimeters 27.0 25.4Position of broadest point centimeters from bottom 10.6 8.1 Flesh coloryellow, orange, cream yellow yellow Flesh color (RHS) RoyalHorticultural Yellow Group Yellow Orange Society Colour Charts 11D Group19B Flesh thickness at stem end centimeters 3.8 3.0 Flesh thickness atmedial centimeters 4.8 3.8 Flesh thickness at blossom end centimeters3.4 2.8 Flesh texture fine, granular, lumpy, stringy stringy stringyFlesh texture soft, firm, brittle firm firm Diameter of blossom scarcentimeters 4.4 3.9 Mature fruit Number of seeds per fruit count 576.8554.5 Weight grams 0.2 0.2 Length centimeters 1.8 2.2 Width centimeters1.2 1.3 Thickness millimeters 1.4 1.6

In tables 2 and 3 the first line shows the trial location and theplanting date, the second column shows the “Number of marketable Fruitper Plant”, the third column shows the “Handle Thickness” in inches 1.5inches from the fruit, the fourth column shows the “Handle Length” ininches, the fifth column “Fruit Length” gives the fruit height ininches, the sixth column gives the “Fruit Width” in inches, column seven“Fruit Weight” is given in pounds as an average or range.

TABLE 2 Freemont, OH June 8 Handle Number of Thickness Marketable(Inches) 1.5 Handle Fruit Fruit Fruit Fruit per inches from LengthLength Width Weight Variety: Plant Fruit (Inches) (Inches) (Inches)(pounds) HMX2738 1 1.25 6  9 11 18 Magic 1 1.25 4 10 12 19 Wand

TABLE 3 South Bend, IN June 9 Handle Number of Thickness Marketable(Inches) 1.5 Handle Fruit Fruit Fruit Fruit per inches from LengthLength Width Weight Variety: Plant Fruit (Inches) (Inches) (Inches)(pounds) HMX2738   2   1   7 10 10 14-22 Magic 1.5 1.5 4.5 10 11 18 WandDeposit Information

A deposit of the pumpkin seed of this invention is maintained byHM.CLAUSE, Inc., 9241 Mace Boulevard, Davis, Calif. 95618 USA. Inaddition, a sample of the hybrid pumpkin seed of this invention has beendeposited with the National Collections of Industrial, Food and MarineBacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY,United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited hybrid pumpkin HMX2738(deposited as NCIMB Accession No. 43504).

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;

4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 CFR 1.807;and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of hybrid pumpkin designated HMX2738,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 43504. 2. A pumpkin plant, a plant partthereof, or a plant cell thereof, produced by growing the seed of claim1, wherein a pumpkin plant regenerated from said plant part or plantcell has all of the physiological and morphological characteristics ofhybrid pumpkin HMX2738 when grown under the same environmentalconditions.
 3. The pumpkin plant part or a plant cell thereof of claim2, wherein the pumpkin part is selected from the group consisting of aleaf, a flower, a fruit, a cell, a rootstock, and a scion.
 4. A tissueculture of regenerable cells produced from the plant or plant part ofclaim 2, wherein a plant regenerated from the tissue culture has all ofthe physiological and morphological characteristics of hybrid pumpkinHMX2738 when grown in the same environmental conditions.
 5. A pumpkinplant regenerated from the tissue culture of claim 4, said plant havingall of the physiological and morphological characteristics of hybridpumpkin HMX2738 when grown under the same environmental conditions,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 43504. 6. A method for harvesting a pumpkinfruit, said method comprising, (a) growing the pumpkin plant of claim 2to produce a pumpkin fruit, and (b) harvesting said pumpkin fruit.
 7. Amethod for producing a pumpkin seed, said method comprising: crossing afirst parent pumpkin plant with a second parent pumpkin plant andharvesting the resultant pumpkin seed, wherein said first parent pumpkinplant and/or second parent pumpkin plant is the pumpkin plant of claim2.
 8. A method for producing a pumpkin seed, said method comprising:self-pollinating the pumpkin plant of claim 2 and harvesting theresultant pumpkin seed.
 9. A method of vegetatively propagating apumpkin plant, said method comprising: (a) collecting a plant part ofthe plant of claim 2 and (b) regenerating a plant from said plant part.10. The method of claim 9, further comprising harvesting a fruit fromsaid plant obtained from step (b) of claim
 9. 11. A plant obtained fromthe method of claim 9, wherein said plant has all of the physiologicaland morphological characteristics of hybrid pumpkin HMX2738 when grownunder the same environmental conditions.
 12. A pumpkin fruit selectedfrom (1) a pumpkin fruit produced from the plant of claim 2, (2) apumpkin fruit produced by the method of claim 6, and (3) a pumpkin fruitobtained from the method of claim
 10. 13. A method of producing apumpkin plant derived from the hybrid pumpkin HMX2738, said methodcomprising: (a) self-pollinating the plant of claim 2 at least once toproduce a progeny plant derived from the hybrid pumpkin HMX2738.
 14. Themethod of claim 13, further comprising the steps of: (b) crossing theprogeny plant derived from the hybrid pumpkin HMX2738 with itself or asecond plant to produce a seed of progeny plant of subsequentgeneration, wherein the second plant is a cucurbita plant; (c) growingthe progeny plant of the subsequent generation from the seed; (d)crossing the progeny plant of the subsequent generation with itself orthe second plant to produce a pumpkin plant derived from the hybridpumpkin HMX2738; and (e) repeating step (b) and/or (c) for at least onegeneration to produce a pumpkin plant derived from the hybrid pumpkinHMX2738.
 15. A method of producing a cucurbita plant derived from thehybrid pumpkin HMX2738, said method comprising, (a) crossing the plantof claim 2 with a second plant to produce a progeny plant, wherein thesecond plant is a cucurbita plant.
 16. The method of claim 15, furthercomprising the steps of: (b) crossing the progeny plant derived from thehybrid pumpkin HMX2738 with itself or the second plant to produce a seedof progeny plant of subsequent generation; (c) growing the progeny plantof the subsequent generation from the seed; (d) crossing the progenyplant of the subsequent generation with itself or the second plant toproduce a cucurbita plant derived from the hybrid pumpkin HMX2738; and(e) repeating step (b) and/or (c) to produce a cucurbita plant derivedfrom the hybrid pumpkin HMX2738.
 17. A pumpkin plant, comprising: asingle locus conversion and otherwise all of the physiological andmorphological characteristics of hybrid pumpkin HMX2738 when grown underthe same environmental conditions, wherein a representative sample ofseed of said hybrid pumpkin plant has been deposited under NCIMB No.43504.
 18. The plant of claim 17, wherein the single locus conversionconfers said plant with herbicide resistance.
 19. The plant of claim 17,wherein the single locus conversion is introduced into the plant byartificial mutagenesis.
 20. The plant of claim 17, wherein the singlelocus conversion is introduced into the plant by a breeding techniqueselected from the group consisting of Zinc finger nuclease (ZFN)technology, oligonucleotide directed mutagenesis, cisgenesis,intragenesis, RNA-dependent DNA methylation, reverse breeding,agro-infiltration, Transcription Activation-Like Effector Nuclease(TALENs), CRISPR/Cas system, engineered meganuclease, re-engineeredhoming endonuclease, and DNA guided genome editing.
 21. A method ofproducing a pumpkin plant, said method comprising, grafting a rootstockor a scion of the hybrid pumpkin plant of claim 2 to another pumpkinplant.
 22. A method for producing nucleic acids, said method comprising:isolating nucleic acids from the plant of claim 2, or a plant part, or aplant cell thereof.
 23. A method for producing a second pumpkin plant,said method comprising: applying plant breeding techniques to the plantor plant part of claim 2 to produce the second pumpkin plant.